cAMP-dependent protein kinase (cAPK), ubiquitous in mammalian cells, is one of the simplest and best understood members of the protein kinase superfamily. The inactive holoenzyme is comprised of two catalytic (C) subunits and a regulatory (R) subunit dimer. cAMP binding to R causes the release of active C. The goals of this grant are to elucidate the structure, function, and conformational diversity of the R subunits. The R subunits are highly dynamic, modular, multifunctional proteins that also target cAPK to specific sites through interactions with A Kinase Anchoring Proteins (AKAPs). The isoform diversity of cAPK is mediated primarily through the R subunits (RI-alpha, RI-beta, RII-alpha, and RII-beta) which have important biological and functional differences. Nevertheless, all share a common architecture with a stable dimerization domain at the N-terminus followed by a variable linker and then two tandem cAMP binding domains. The dimerization domain is also the docking site for AKAPs. During this past granting period our understanding of the structure of this protein family grew substantially by solving structures of a monomeric RII-beta and the RI-alpha dimerization domain. In parallel, using time-resolved fluorescence anisotropy (TRFA), the principal investigator began to map the dynamic features of RI-alpha in solution. Major goals during this next granting period are three fold. First will be to solve a structure of a holoenzyme complex. This structure is essential if we are to understand the molecular switching mechanisms that R undergoes as it shuttles between its cAMP bound conformation and its holoenzyme conformation. The principal investigator will begin with monomeric forms of RI-alpha bound to C and then move to full-length holoenzyme complexes. In parallel the principal investigator will crystallize holoenzyme complexes with RII-beta. The cAMP binding domains of R are ancient, highly conserved modules that mediate the biological response of cAMP. The structures of RII-beta compared with RI-alpha and CAP allow to better understand the buried and conserved cAMP binding cassette. The principal investigator will continue to probe this important signaling module by crystallizing it with the cAMP antagonist, Rp-cAMP, and with site specific analogs of cAMP. In addition she will use mutagenesis to probe the extended network of interactions that link the cAMP binding site to distant parts of the protein. In parallel with crystallization, the principal investigator will use solution methods to characterize the global and local dynamic properties of the R subunits. In addition to using the endogenous fluorescence of RII-beta and TRFA with labeling of single site cysteines in RI-alpha, the principal investigator has begun to comprehensively map the R subunits in their free, cAMP-bound, and holoenzyme conformations using H/D exchange coupled with mass spectrometry. Mutant forms of RI-alpha and RII-beta will also be characterized.